Immunological detection of serpin in the fall webworm, Hyphantria cunea and its inhibitory activity on the prophenoloxidase system

We previously identified a serine type protease inhibitor (serpin) cDNA, us ing PCR-based differential display, in the fall webworm which mas up-regula ted following a bacterial challenge (Shin et al,, 1998), The serpin cDNA wa s inserted into an expression vector and the serpin protein was expressed i n Escherichia coli. In order to investigate the action of serpin in vivo, w e examined the concentration of serpin protein in the larvae of Hyphantria cunea by Western blot analysis using a polyclonal antibody raised in a rabb it injected with recombinant serpin, H, cunea serpin was found mainly in th e plasma with a molecular mass of 56.6 kDa on SDS-PAGE followed by Western blot analysis, The concentration of serpin in the plasma was slightly incre ased following bacterial challenge, A new 50.5 kDa (approx.) band was detec ted post E, coli and distilled water injection. Both E, coli and distilled water injection induced increased phenoloxidase (PO) activity in the plasma , although E, coli injection produced a larger increase in activity, Hyphan tria serpin probably participates in negative regulation of the prophenolox idase (proPO) cascade. Recombinant serpin inhibits PO activity in the hemoc yte lysate fraction activated by LPS, There is a similarity between the P-2 -P-2' region (NKFG) of the serpin reactive site loop and the S-2-S-2' regio n (NRFG) of the insect proPO maturation site, This indicates a form of comp etitive inhibition of serpin against a protease involved in the activation of proPO. A tyrosine residue in the P-11 region of serpin, which is conserv ed in the S-11 regions of all known proPOs maturation sites, provides furth er support for this hypothesis.